Over the last year, the TCD Section has focused on three major projects. One project focused on the roles of Notch and GATA3 in T cell differentiation in the thymus. This line of investigation arose out of our earlier studies showing that Notch potentiates TCR signaling in positive selection and in the generation of mature CD4 and CD8 T cells. Since then a primary focus of the lab has been to identify the downstream mediators linking the TCR and Notch signaling pathways. We explored the possibility that GATA3 may function in this capacity. We had observed that Notch and GATA3 operate similarly at successive stages of thymocyte development. Moreover, Notch was reported to directly bind the GATA3 locus and regulate GATA3 in peripheral T helper 2 (Th2) responses. These findings prompted us to ask whether Notch facilitates TCR signaling and thymocyte selection via the regulation of GATA3 function and/or expression. To facilitate these studies, we obtained mice with conditional gene deletion of GATA3 or GATA3 transgenes expressed in immature thymocytes. Notably, forced GATA3 expression in Notch signaling mutant (Presenilin-deficient) mice rescued development of CD4 T cells, suggesting that Notch and GATA3 could function cooperatively to promote thymocyte selection. These analyses of the effects of GATA3 mutation led to some unexpected findings and a broader investigation into how GATA3 functions in T cell development. The original report on GATA3-deficient thymocytes demonstrated that GATA3 was required for the generation of CD4 T cells. Because the authors failed to find any phenotypic or signaling defects in the precursor thymocytes, they concluded that the failure to develop CD4 T cells was due to a post-maturational block occurring after CD4/CD8 T lineage commitment. We found to the contrary that GATA3-deficiency and GATA3 overexpression reciprocally modulated in vivo indicators of positive selection before lineage commitment. Moreover, in vitro assays revealed that TCR signal transduction was attenuated in GATA3-deficient thymocytes during and even prior to thymocyte selection. While it was known that GATA3 expression was directly related to the strength of TCR stimulation, our results suggested that the reverse was also true; GATA3 promotes TCR signaling. From these data, we propose that TCR and GATA3 operate in a positive reinforcing loop with each amplifying the effects of the other. Since MHC2 generates stronger/ sustained TCR signals, this amplifying loop would generate more GATA3 in MHC2-selected CD4 thymocytes, explaining the preferential loss of CD4 T cells with GATA3-deficiency. It had been reported that GATA3 plays no role in CD4/CD8 T lineage commitment. We generated experimental models to better address this question. Also, we had collaborated with R. Bosselut (NCI) to determine how GATA3 interacts with a second transcription factor that is essential for CD4 T lineage commitment, ThPOK. In GATA3-deficienct TCR transgenic or MHC1-deficient mice, we demonstrated that MHC2-selected thymocytes could be re-directed from the CD4 to CD8 lineage. Although we found that GATA3 binds the Thpok locus and acts to trigger ThPOK expression, a ThPOK transgene could not rescue CD4 development in GATA3-deficient mice. The latter result can be explained by our current view that GATA3 participates both in TCR signaling during positive selection and in lineage commitment. In another project, we have investigated the function of the intracellular domain of preTCR alpha chain (pTa) in preTCR signaling and early T cell development. We used a knock-in gene targeting approach to replace the endogenous locus with a truncated gene encoding a pTa protein lacking the intracellular domain. Truncation of the pTa cytoplasmic tail impairs the efficiency of TCR beta allelic exclusion, cell cycle at the proliferative stage, and the generation of more mature thymocytes. To facilitate these studies, we introduced the pTa tailless knock-in mutation into mice that can only express one TCR, the preTCR. Surface levels and internalization of the preTCR were normal allowing an objective evaluation of the contribution of the tail to preTCR signaling. Single cell assays for signal transduction in response to TCR stimulation reveled significant impairment with tailless pTa. In vivo indicators of preTCR signaling were also diminished. Collectively, these data suggest that the pTa cytoplasmic domain serves to potentiate preTCR signaling in early T cell development. In a third project, we focused on the question of how Notch functions in the activation and differentiation of peripheral T cells. Studies from many groups investigating the role of Notch function in CD4 T helper cell differentiation have yielded conflicting results. Notch has been described as instructive for the differentiation of Th1, Th2, Th17, and regulatory T cells, but it is difficult to understand how Notch could direct commitment to all of these distinct lineages. A collaborative investigation of Notch function in Th2 differentiation and our previous work showing that Notch enhances TCR signaling in developing thymocytes led us to focus on the effects of Notch signaling in the initial stages of peripheral T cell activation. We used mouse models bearing TCR with defined peptide specificities and conditional mutations in Notch signaling. Also, we generated the first mouse model with a conditional deletion of Notch ligand, DLL4, in dendritic cells (DC). Antigen-specific T cells were used in a highly defined in vitro experimental system with purified normal or mutant antigen presenting cells (APC) from spleen or with fibroblast lines that we engineered to express Notch ligands in combination with other costimulatory receptors. These experimental approaches demonstrated that Notch signaling promotes the initial stages of CD4 T cell activation; enhancing metabolism, proliferation, and IL-2 secretion. CD4 T cell dependence upon Notch varied inversely with antigen dose, with the most significant differences occurring when peptide was low. The same results were obtained whether Notch ligand DLL4 was absent from the APC, or Notch signaling was disrupted in T cells. These data strongly indicated that Notch signaling in CD4 T cells facilitates antigen dependent activation. In all cases, Notch signaling affected the frequency of responding T cells rather than the magnitude of the response generated by a single cell. Notch ligand DLL4 conferred fibroblast APC with superior T cell stimulatory activity, and fulfilled the criteria of a classic costimulatory molecule by providing the T cell with a second signal from the APC and by reducing the amount of peptide required to activate a naive CD4 T cell. An unexpected result arising from this study was that the costimulatory activity of DLL4/Notch was dependent on another molecule (expressed by APC) that is costimulatory for T cell activation, CD80. Without signaling through the TCR or CD28 (the receptor for CD80), DLL4 had no effect on T cell activation. Thus, Notch- and CD28-mediated costimulation worked synergistically to set the threshold for activation as well as inducing the calcium, NFkB, and RAS/MAPK pathways downstream of the TCR. To our knowledge, this is the first example in which another receptor has been demonstrated to costimulate CD28-mediated costimulation in CD4 T cells. These findings also may help to resolve some controversies in the field. Since Notch signaling promotes the initial stages of T cell activation and IL-2 production in naive CD4 T cells, this function accounts for Notch effects on Th2 differentiation that we previously demonstrated, but perhaps also for other lineages since IL-2 plays an essential role in the differentiation of most Th lineages.